Protein tyrosine kinases and protein tyrosine phosphatases are involved in abscisic acid-dependent processes in Arabidopsis seeds and suspension cells

Abstract

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

Figure 1.

ABA induction of RAB18 gene expression is mediated by PTP activities in Arabidopsis suspension cells. Northern-blot analysis of total RNA (10 μg) from cells incubated for 3 h with ABA (10 μm) and various PTP inhibitors is shown. A, Phenylarsine oxide (1 to 10 μm). B, Dephostatin (100 μm). C, NAP (200 μm). Control cells were treated with DMSO alone. Ethidium bromide staining of 25S and 18S rRNAs is shown as the control.

Figure 2.

PTKs are involved in the signal transduction pathway leading to RAB18 gene expression in Arabidopsis suspension cells. Northern-blot analysis of total RNA (10 μg) from cells incubated for 3 h with ABA (10 μm) and various PTK inhibitors is shown. A, Genistein (100 μm) and daidzein (100 μm). B, Erbstatin (100 μm). C, Tyrphostin A23 (100 μm) and tyrphostin A25 (100 μm). D, Lavendustin A (20 μm) and tyrphostin AG490 (100 μm). Ethidium bromide staining of 25S and 18S rRNAs is shown as the control.

Figure 3.

Effects of PAO and genistein on stomatal closure induced by ABA in Arabidopsis. Preopened stomata were exposed to ABA (10 μm), PAO (10 μm), ABA (10 μm) and PAO (10 μm), genistein (100 μm), and ABA (10 μm) and genistein (100 μm) for 3 h. The stomata were preincubated with PAO or genistein for 30 min before adding ABA. Values represent means of 70 measurements. Each experiment was repeated three times. Error bars indicate se.

Figure 4.

ABA modulates protein abundance in Arabidopsis seeds. Seeds were incubated for 2 d in water or in ABA (10 μm). An equal amount of each total protein extract (1.25 mg) was loaded onto each gel. The results shown are representative of at least three repetitions. 2D gels of total proteins were stained with Coomassie Brilliant Blue. MM, Molecular mass markers. A, Coomassie Brilliant Blue-stained 2D gel of total proteins from seeds imbibed for 2 d in water. B, Coomassie Brilliant Blue-stained 2D gel of total proteins from seeds imbibed for 2 d in the presence of ABA. Windows a and b locate the areas of the gel where the protein abundance is modulated by ABA.

Figure 5.

Tyr phosphorylation levels of Arabidopsis seed proteins are regulated by ABA. Total proteins were extracted from seeds and separated on 2D gels. Tyr phosphorylated proteins were detected by western blot with an anti-phosphotyrosine antibody. MM, Molecular mass markers. A, Immunoblot of 2D gel of total proteins from seeds imbibed for 2 d in water. B, Immunoblot of 2D gel of total proteins from seeds imbibed for 2 d in the presence of ABA (10 μm). The Tyr phosphorylation level of proteins labeled with diamonds was unaffected by ABA. The indicated portions of the immunoblots are reproduced in C. C, Enlarged windows (a–d) of immunoblots as shown in A and B for control (left) and ABA-treated (right) seeds. The proteins phosphorylated on Tyr residues in response to ABA are squared. Tyr residues of proteins shown in circles are dephosphorylated in the seeds after ABA treatment. These proteins (spots A–S) were analyzed by MALDI-TOF-TOF MS (Table I).

Figure 6.

Effect of genistein on the Tyr phosphorylation induced by ABA. Seeds were imbibed with 0.1% DMSO (Control), 100 μm genistein (Genistein), 10 μm ABA (ABA), or both genistein and ABA (Genistein+ABA) for 2 d. Total proteins were extracted from seeds and separated on 2D gels. Tyr phosphorylated proteins were detected by western blot with PY20 anti-phosphotyrosine antibody, and spots showing variations in ABA-dependent Tyr phosphorylation were quantified. A, Close-up view of the 11 spots corresponding to proteins whose Tyr phosphorylation is induced by ABA. B, Quantitation of the spot intensities. Images were quantitated as described in “Materials and Methods.” The results are means of three experiments.